The overall objective of this project is to determine the factors regulating cardiac adenosine formation and its interstitial concentration. We have developed a technique for calculating interstitial fluid (ISF) adenosine concentration using the indicator dilution technique. We will compare our calculated ISF adenosine with the adenosine concentration measured in epicardial transudates. We will also extend the use of the indicator dilution techniques to the blood perfused heart. In addition, we will use tracer techniques to study nucleotide degradation in in situ coronary endothelial cells. Another objective of this project is to make use of oxygen NMR to test the hypothesis that most adenosine formation is the result of the activity of cytosolic 5' nucleotidase, regulated by the levels of adenine nucleotide compounds and inorganic phosphate. To do this we will take advantage of the fact that the activity of the purified enzyme has a biphasic relationship to the adenine nucleotide phosphorylation potential because it is inhibited by high inorganic phosphate concentration. We will progressively lower the phosphorylation potential in isolated hearts by progressively decreasing the oxygen supply/demand ratio. We will simultaneously measure the phosphorylation potential via oxygen NMR and measure adenosine release into the venous effluent. If our hypothesis is correct, adenosine release should bear a biphasic relationship to the phosphorylation potential. Having obtained this plot of adenosine release vs. phosphorylation potential, we will try other interventions which either increase or decrease adenosine release. If cytosolic 5'nucleotidase is regulating adenosine formation, then the effect of each intervention on adenosine release should be predictable from its effect on phosphorylation potential. Because some of the above interventions cause directionally similar changes in phosphorylation potential and oxygen consumption (MVO2) whereas others cause inverse changes, we will be able to separate the effect of MVO2 from that of phosphorylation potential on adenosine release. We suspect that some interventions (acetylcholine, thrombin, or caffeine infusion) increase adenosine release via a receptor-mediated mechanism, since they do not appear to decrease the oxygen supply/demand ratio. In this case adenosine release should not be predicated by the change in phosphorylation potential.